Method for the treatment or prevention of osteoarthritis

ABSTRACT

The present invention is in the field of medicine and provides means and methods for the treatment, prevention or amelioration of osteoarthritis. More in particular, it provides a peptide for use in the treatment, amelioration or prevention of osteoarthritis, wherein the peptide consists of an amino acid sequence according to SEQ ID NO: 18 or an analogue thereof, wherein the analogue is a peptide consisting of an amino acid sequence according to formula 1 (SEQ ID NO: 29), or a fragment thereof wherein the fragment consists of at least 10 consecutive amino acids of SEQ ID NO: 18 or an amino acid sequence according to formula 1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/092,680, filed Oct. 10, 2018, which is a national phase entry under35 U.S.C. § 371 of International Patent Application PCT/EP2017/057720,filed Mar. 31, 2017, designating the United States of America andpublished in English as International Patent Publication WO 2017/178253on Oct. 19, 2017, which claims the benefit under Article 8 of the PatentCooperation Treaty to European Patent Application Serial No. 16165106.2,filed Apr. 13, 2016, the disclosure of each of which is herebyincorporated herein in its entirety by this reference.

FIELD OF THE INVENTION

The present invention is in the field of medicine and provides means andmethods for the treatment, prevention or amelioration of osteoarthritis.

BACKGROUND

Osteoarthritis (OA) is a type of joint disease that results frombreakdown of joint cartilage and underlying bone, combined with anoverall joint pathology including synovial inflammation and -fibrosisand meniscus pathology. The most common symptoms are joint pain andstiffness. Initially, symptoms may occur only following exercise, butover time may become constant. Other symptoms may include jointswelling, decreased range of motion, and when the back is affected by OAweakness or numbness of the arms and legs may present. The most commonlyinvolved joints are those near the ends of the fingers, at the base ofthe thumb, neck, lower back, knees, and hips. Joints on one side of thebody are often more affected than those on the other. Usually theproblems come on over years. It can affect work and normal dailyactivities. Unlike other types of arthritis, only the joints aretypically affected.

Causes include previous joint injury, abnormal joint or limb developmentabnormal alignment of joints and inherited factors. Risk is greater inthose who are overweight, have one leg of a different length, and havejobs that result in high levels of joint stress. Osteoarthritis isbelieved to be caused by mechanical stress on the joint and low gradeinflammatory processes. It develops as cartilage is lost with eventuallythe underlying bone becoming affected. The subchondral bone is alsothought to be crucially involved in the etiology of the disease. As painmay make it difficult to exercise, muscle loss may occur. Diagnosis istypically based on signs and symptoms with medical imaging and othertests occasionally used to either support or rule out other problems.Unlike in rheumatoid arthritis, which is primarily an inflammatorycondition, the joints do not typically become hot or red.

Treatment includes exercise, efforts to decrease joint stress, supportgroups, joint lubrication and (local) pain medications. Efforts todecrease joint stress include resting and the use of a cane. Weight lossmay help in those who are overweight. Pain medications may includeparacetamol (acetaminophen). If this does not work, orally administeredNSAIDs such as naproxen may be used, or locally administeredcorticosteroids (eg triamcinolonacitonide) may be used, but thesemedications are associated with greater side effects. Opioids if usedare generally only recommended short term due to the risk of addiction.If pain or movement restriction interferes with normal life despiteother treatments, joint replacement surgery may help. An artificialjoint, however, only lasts a limited amount of time and total jointreplacement surgery is associated with severe complications likeosteomyelitis. Outcomes for most people with osteoarthritis are goodafter total joint replacement.

OA is the most common form of arthritis with disease of the knee and hipaffecting about 3.8% of people as of 2010. Among those over 60 years oldabout 10% of males and 18% of females are affected. It is the cause ofabout 2% of years lived with disability. In Australia about 1.9 millionpeople are affected, and in the United States about 27 million peopleare affected. Before 45 years of age it is more common in men, whileafter 45 years of age it is more common in women. It becomes more commonin both sexes as people become older.

While OA is a degenerative joint disease that may cause gross cartilageloss and morphological damage to other joint tissues, more subtlebiochemical changes occur in the earliest stages of OA development. Thewater content of healthy cartilage is finely balanced by compressiveforce driving water out & swelling pressure drawing water in, supportedby a distinct osmotic tissue pressure. Collagen fibers exert thecompressive force, whereas the Gibbs-Donnan effect & cartilageproteoglycans create osmotic pressure which tends to draw water in.

However, during onset of OA, the collagen matrix becomes moredisorganized and there is a decrease in proteoglycan content withincartilage. The breakdown of collagen fibers results in a net increase inwater content. This increase occurs because whilst there is an overallloss of proteoglycans (and thus a decreased osmotic pull), it isoutweighed by a loss of collagen. Without the protective effects of theproteoglycans, the collagen fibers of the cartilage can becomesusceptible to degradation and thus exacerbate the degeneration.Inflammation of the synovium (joint cavity lining) and the surroundingjoint capsule can also occur, though often mild (compared to what occursin rheumatoid arthritis).

Changes in the articular cartilage and articular chondrocytes thatcharacterize OA resemble the cellular developmental process drivingskeletal development by endochondral ossification. The analogy betweenendochondral ossification and OA progression has been widely recognized.Many of the cartilage-degrading enzymes that are excreted byhypertrophic chondrocytes in the growth plate are also central inprogression and worsening of the OA condition. Also, well-known pathwayscontrolling chondrocyte differentiation in the growth plate (RUNX2,COL10A1, ALP) are found active or deregulated in OA articularchondrocytes as well.

Other structures within the joint can also be affected. The ligamentswithin the joint become thickened and fibrotic and the menisci canbecome damaged and wear away. Menisci can be completely absent by thetime a person undergoes a joint replacement. New bone outgrowths, called“spurs” or osteophytes, can form on the margins of the joints, possiblyin an attempt to improve the congruence of the articular cartilagesurfaces in the absence of the menisci. The subchondral bone volumeincreases and becomes less mineralized (osteoporotic/osteopenic). Alsobone marrow lesions occur as a result of osteoarthritic changes in thesubchondral bone. All these changes can cause problems in functioningand mechanical support for the overlying cartilage layer. The pain in anosteoarthritic joint has been related to thickened synovium andsubchondral bone lesions.

Biochemically, OA is characterized by synthesis of extracellular matrix(ECM)-degrading enzymes, such as aggrecanases (a disintegrin andmetalloproteinase with trombospondine motifs (ADAMTSs)) and matrixmetalloproteinases (MMPs), resulting in the active breakdown of thecartilage tissue matrix. The analogy between endochondral ossificationand OA progression has been recognized and many of the cartilagedegrading and mineralization enzymes that are secreted by hypertrophicchondrocytes in the growth plate are also crucially involved in OA.

Notwithstanding the progress made in the understanding of diseasemechanisms, established and experimental treatment of OA is mainlysymptomatic by alleviating pain and interfering with the cartilagedegenerative processes to postpone total joint replacement.

SUMMARY OF THE INVENTION

The invention relates to a peptide derived from the full length humanprotein called bone morphogenetic protein-7 (BMP-7) for use in thetreatment, amelioration or prevention of osteoarthritis. More inparticular, the invention relates to a peptide for use in the treatment,amelioration or prevention of osteoarthritis wherein the peptideconsists of an amino acid sequence according to SEQ ID NO: 18 or ananalogue thereof, wherein the analogue consists of an amino acidsequence according to formula 1, or a fragment thereof wherein thefragment consists of at least 10 consecutive amino acids of SEQ ID NO:18 or an amino acid sequence according to formula 1.

Formula 1: (SEQ ID NO: 29) P   K   P   S   S  X1  P  X2 X3  L  X4 X5  I X6  V  X7 X8 X9  D  X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21  M  V   V X22 X23  S   G   S  X24,

wherein;

X1=A or V

X2=T or D or E

X3=Q or D or K or S

X4=N or D or E or H

X5=A or D or S

X6=S or I or L or M or F or Y or E or H or K or Q or R or D

X7=L or E or K or T or M or R

X8=Y or A or D or E or H or K or S or T

X9=F or H or A or D or E or K or Q or R or Y

X10=D or I or L or E or N or S or T

X11=S or D or E or N or R

X12=S or D or E or K or N or T

X13=N or E or Q or R

X14=V or A or D or N or R or T or M or Y or H

X15=I or A or D or E or K or N or Q or R or S or T or V

X16=L or A or E or K or Q

X17=K or D or E or G or Q or R or W

X18=K or A or D or E or H or N or P or Q or S or T or I or V or M

X19=Y or D or E or H or K or Q or I or R

X20=R or D or E or K or N or S

X21=N or D

X22=R or E or S or D or K or Q or L

X23=A or E or D or S

X24=H or R.

Wherein X [#] indicates an amino acid position in the human BMP-7protein that is variable.

DETAILED DESCRIPTION OF THE INVENTION

Osteoarthritic chondrocytes display a typical phenotype that ischaracterized by decreased expression of chondrogenic genes SOX9,COL2A1, ACAN and BAPX1/NKX3.2 and an increased expression ofhypertrophic genes RUNX2, COL10A1, ALP, and an increased expression ofgenes encoding cartilage matrix degrading enzymes MMP13, ADAMTS5 andinflammatory genes COX-2 and IL-6 (FIG. 1). We show herein that aprotein called bone morphogenetic protein-7 (BMP-7, also called OP-1) iscapable of rescuing the OA phenotype (FIG. 1). Ours and earlier studies[22,23] addressing the disease-modifying properties of BMP-7 show thatit decreases MMP13 expression in IL-1β-exposed chondrocytes, stimulatesproteoglycan synthesis in OA chondrocytes, counteracts inflammatorycytokines (e.g. IL-1β) and induces an anabolic response in healthychondrocytes. Intra-articular administration of BMP-7 protects againstOA development and delays progression of OA in rats. A phase-1 clinicaltrial has been completed in OA patients and reported no serious adverseevents after intra-articular injection of BMP-7. In accordance withthis, the data reported herein unveil that BMP-7 actively suppresses the(OA) chondrocyte hypertrophic phenotype (FIG. 1).

Despite these promising results, intra-articular use of full-lengthrecombinant human BMP-7 for OA-treatment may not be suitable forclinical use. Pre-clinical testing showed that weekly intra-articularBMP-7 injections were necessary to acquire a relevant result. This highfrequency of intra-articular injections is not acceptable for clinicaluse due to risk of septic arthritis and patient discomfort. While asolution to prevent frequent intra-articular injections would encompassthe encapsulation of BMP-7 in an intra-articular release system forlong-term controlled release, retaining bioactivity of BMP-7 will posean enormous challenge due to the denaturing conditions that generallyapply for the production process of currently existingcontrolled-release systems. OA synovial fluid, in which BMP-7 is likelyto be delivered, is a harsh hydrolytic and proteolytic environment thatis expected to cause rapid degradation of the administered BMP-7.Finally, production of GMP-grade BMP-7 is technologically demanding withaccompanying high costs.

To safeguard and enable the clinical use of the highly favourablecharacteristics of BMP-7 activity for OA-treatment, we sought for BMP-7molecular mimics that are better compatible with the harsh OA synovialfluid environment and can potentially be incorporated in intra-articularmolecular release systems for long-term release. For that reason we setout to prepare a set of overlapping 20-mer peptides (table 1) thatcollectively cover the entire mature BMP-7 polypeptide of 139 aminoacids (SEQ ID NO: 27, table 3). The results confirmed earlier findings[20, 21] in that none of the peptides mimicked the potential of BMP-7 torescue the OA phenotype. In more detail, all peptides shown in table 1were tested for expression of genes IL-6, ADAMTS5, COL10A, ALP,BAPX1/NKX3.2 and RUNX2 and found to evoke pro-hypertrophic,pro-mineralizing, pro-katabolic and pro-inflammatory responses. This wasnot unexpected, since pro-mineralizing/pro-osteogenic peptides have beendescribed from BMP-2 [20, 32] and BMP-7 [21, 31].

Surprisingly, however, when we synthesized overlapping 20-mer peptidesfrom a region constituted by amino acids 93-139 (SEQ ID NO: 1) of themature human BMP-7 and replaced the Cysteine residues in that regionwith Serine residues (SEQ ID NO: 28, table 3), we found that peptidesfrom the region formed by amino acids 100-139 were active aspro-chondrogenic and anti-inflammatory agents. A selection of peptidesfrom this region was identified before as osteogenic agents, useful forinducing bone formation [31]. This region of amino acids 100-139 of thehuman BMP-7 polypeptide is further referred to herein as “region B”.

Table 2 shows the results of expression analysis of several genes in thepresence of several peptides for use according to the invention. Furtherdetails are provided in the Examples section.

In more detail; peptides shown in table 2 were tested for expression ofgenes IL-6, ADAMTS5, COL10A, ALP, BAPX1/NKX3.2 and RUNX2. Much to oursurprise, we found that peptides with an amino acid sequencecorresponding to amino acids 100-139 of BMP-7 or fragments thereofevoked the sought-after BMP-7-mimicking activity. This means that theyinduced a pro-chondrogenic and anti-inflammatory response. This wasunexpected, since so far only pro-mineralizing and pro-osteogenicpeptides have been described from BMP-2 [20] and BMP-7 [21].

TABLE 3 Amino acid sequence of mature BMP-7, Cysteine and Serineresidues underlined. Name Amino Acid Sequence SEQ ID NO: Mature BMP-7STGSKQRSQN RSKTPKNQEA LRMANVAENS 27 SSDQRQA C KK HELYVSFRDL GWQDWIIAPEGYAAYY C EGE  C AFPLNSYMN ATNHAIVQTL VHFINPETVP KP CC APTQLN AISVLYFDDSSNVILKKYRN MVVRA C G C H Mature BMP-7 STGSKQRSQN RSKTPKNQEA LRMANVAENS28 with Cysteine SSDQRQA S KK HELYVSFRDL GWQDWIIAPE replaced by GYAAYY SEGE  S AFPLNSYMN ATNHAIVQTL Serine VHFINPETVP KP SS APTQLN AISVLYFDDSSNVILKKYRN MVVRA S G S H

Consecutive and overlapping peptides from region B caused significantlydecreased expression of OA chondrocyte phenotypic markers IL-6, ADAMTS5,COL10A, ALP and RUNX2 (annotated as “+” in table 2, as well as increasedexpression of chondrogenic marker BAPX1/NKX3.2 (annotated as “+” intable 2) in OA articular chondrocytes (Table 2).

Since 10-mer peptides from this region were previously shown to beactive in increasing osteoblast proliferation and promoting osteoblastcalcium deposition [21], it may safely be assumed that peptides of thislength will also be useful in the present invention. In anotherembodiment of the invention, the peptides may advantageously be longer,such as 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acidslong. Longer peptides may also be employed, such as peptides of morethan 21 amino acids such as more than 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, or more than 38, such as 39 or 40 aminoacids.

In one aspect, the invention therefore relates to a peptide for use inthe treatment, amelioration or prevention of osteoarthritis, wherein thepeptide comprises an amino acid sequence of at least 10 consecutiveamino acids selected from the sequence according to SEQ ID NO: 18. Thisis deduced from the results shown in table 2.

Our experiments also revealed that the peptide has a maximum length of40 amino acids or thereabout. The peptide for use according to theinvention is preferably fully contained in the C-terminal 40 amino acidsof BMP-7 (SEQ ID NO: 18). Every peptide that had an additional aminoacid N-terminal of position 100 (the N-terminus of SEQ ID NO: 18) lostits activity in the assays as employed herein (table 2, see for examplepeptides with amino acid sequence according to SEQ ID NO: 2, 3, 4 and5).

Of course, this description should not be interpreted so narrowly thatthere is no variation allowed in the peptides for use in the inventionas described above. The skilled person is aware of the fact that theconformation of the peptide is conserved even when one or more, such astwo, three, four or even five amino acids are changed, in particularwhen these changes relate to conservative amino acid substitutions.These peptides are known in the art as homologous peptides. Hence, theterm “homologues” or “analogues” as used herein refers to peptides thatretain their activity but differ with respect to their amino acidsequence. Homologues or analogues can be 75% identical with thesequences according to SEQ ID NO: 18 or fragments thereof or more, suchas 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99% identical.

As used herein, the degree of identity between two or more amino acidsequences is equivalent to a function of the number of identicalpositions shared by the sequences (i.e., % identity=number of identicalpositions divided by the total number of positions×100), excluding gaps,which need to be introduced for optimal alignment of the two sequences,and overhangs. The comparison of sequences and determination of percentidentity between two or more sequences can be accomplished usingstandard methods known in the art. For example, a freewareconventionally used for this purpose is “Align” tool at NCBI recoursehttp://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq

In a preferred embodiment, the alignment of two sequences is to beperformed over the full length of the polypeptides.

As used herein, the term “may” encompasses the word “can,” and the term“may be” encompasses the words “is” or “are,” depending on context.Furthermore, presence of the word “may” is intended to explain optionsfor practicing or implementing the disclosure, without limitation.

We concluded that human BMP-7-derived peptides as described herein areable to mimic the favourable characteristics of the full-length humanBMP-7 protein on the OA chondrocyte phenotype.

We have therewith determined the location and nature of the OAphenotype-suppressive action of BMP-7, whereas peptides from otherregions in BMP-7 displayed no activity or pro-mineralizing/-hypertrophicactions. The bioactive potency of the candidate peptides wasunexpectedly high. Independent of the tested concentration (1000, 100,10 and 1 nanoMolar (nM) were tested), almost all region-B peptidesinduced similar fold-change gene expression magnitude differences in thescreening, whereas a random control peptide with amino acid sequenceSFILKKVLYDRVNDSANIYS (SEQ ID NO: 49) did not.

BMP-7 has unique OA chondrocyte-phenotype suppressive actions (FIG. 1).This action is most pronounced when a BMP-7 concentration is used around1 nM. BMP-7 concentrations higher than 1 nM (eg 10 nM or 100 nM) haveopposite and negative effects on the chondrocyte phenotype and thusunfavorably cause increased levels of hypertrophy, increasedmineralisation, increased expression of cartilage breakdown genes andincreased expression of inflammatory genes. Without wanting to be boundby theory, we speculate that BMP-7 incorporates a dual activity that isdependent on the concentration of BMP-7.

Surprisingly and in contrast with the full-length BMP-7 polypeptide, the20-mer peptides according to SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14,15, and 17 efficiently suppressed the OA chondrocyte phenotype,independent of the tested concentration (1000, 100, 10 and 1 nM weretested). With this we have unexpectedly identified the region comprisingthe favorable OA phenotype-suppressive bioactivity of BMP-7, whilepeptides from other regions in BMP-7 displayed pro-mineralizing,pro-hypertrophic, pro-katabolic and pro-inflammatory actions.

Next to the 24 hours to which OA chondrocytes were exposed to individualpeptides, the OA phenotype-suppressive action of the candidate peptideswas highly effective when peptides were supplemented to OA chondrocytecultures every second until day 10 in culture. Surprisingly, the OAphenotype-suppressive action of the peptides for use according to theinvention even lasted up to 8 days in culture after a single initial48-hours exposure.

The highly potent bioactivity at nanomolar-range concentration is atleast similar to full-length mature BMP-7 and is a therapeuticallyimportant determinant, showing that the peptides for use in theinvention are indeed powerful BMP-7 mimics for OA therapeutical use.

In addition to the action of the peptides for use in the invention on OAchondrocytes, the candidate peptides displayed biologically similaractions in an in vitro model for chondrogenic differentiation.Remarkably, eight days of single exposure, and even more pronounced withcontinuous peptide exposure of differentiating chondroprogenitor cells(ATDC5), solidly decreased the expression of hypertrophy andmineralization markers, while aggrecan expression (healthy chondrocytemarker) and BAPX1/NKX3.2 expression (anti-hypertrophy factor) was evenfurther increased under continuous exposure to the peptides for useaccording to the invention.

Taken together, the peptides for use according to the invention lowerthe inflammatory OA chondrocyte phenotype, while at the same timesupporting a pro-chondrogenic action on OA chondrocytes and healthychondrocytes. In contrast to the full-length mature BMP-7 protein,peptides may advantageously be used in therapy because they are ingeneral less susceptible for conformational and enzymatic inactivation.Peptides can be biochemically fine-tuned to increase their stability andactivity; functionalized for carriers; are much smaller and thussuitable for incorporation in application-dedicated release systems forlong-term intra-articular release.

Variants of the peptides as described above may be synthesized that aremore resistant to degradation in proteolytic degradation in synovialfluid. Also, variants may be prepared that are more conformationallyrestricted and thus be more bioactive as compared to their original leadsequences. Such is well within reach for a skilled person, and requiresonly routine techniques now that the region B has been identified as theactive region for the anabolic activity of BMP-7.

Such variants may include linear peptides, linear retro inversopeptides, retro-inverso peptides, cyclic peptides, mono-looped peptides,and two-looped peptides. CLIPS technology enables the routine productionof such peptides (http://www.pepscan.com/therapeutics/clips-platform).Two-looped peptides may contain two identical peptides as describedherein or two different peptides as described herein.

An L-peptide has three analogue sequences built from L and D aminoacids: the D-enantiomer or inverso-peptide with the same sequence, butcomposed of D-amino acids and a mirror conformation; the retro-peptide,consisting of the same sequence of L amino acids but in reverse order;and the retro-inverso or D-retro-enantiomer peptide, consisting ofD-amino acids in the reversed sequence. While the L-peptide and itsD-enantiomer are mirror structures of each other, the L-retro-peptide isthe mirror image of the D-retro-inverso-peptide. On the other hand, theL-peptide and the D-retro-inverso-peptide share a similar arrangement ofside-chains, although their carboxyl and amino groups point in opposingdirections. For small peptides that do not depend on a secondarystructure for binding, an L-peptide and its D-retro-inverso-peptide islikely to have a similar binding affinity with a target L-protein.

Peptides may be more active when they are looped, such as cyclicpeptides. Such peptides may also be more stable and resistant toproteolytic degradation.

Such peptides and polypeptides may be more stable in an environmenthostile to linear polypeptides, such as a hydrolytic or proteolyticenvironment. In particular for therapeutic purposes, it may beadvantageous to use more stable peptides, such as the ones mentionedabove. Hence, the invention also relates to the use of peptides selectedfrom the group consisting of linear peptides, linear retro-inversopeptides, retro-inverso peptides, cyclic peptides, mono-looped peptides,and two-looped peptides.

Ways to render a peptide less susceptible to degradation are known inthe art, such as the inclusion of one or more non-natural amino acids,such as the D-enantiomer of an L-amino acid and the retro orientation ofthe peptide backbone in the retro-inverso variant. These peptides arenon-natural, hence natural proteases are not able to cleave them.

Means and methods for increasing the bioactivity of the peptides for useaccording to the invention are also known in the art. Examples of suchtechniques are cyclization and looping of the peptides, which provides amore constraint conformational context of essential amino acid residues.

Patent application US2006/0058231 discloses BMP-7 variants with improvedproperties. This document is hereby incorporated by reference.US2006/0058231 discloses that some amino acids of BMP-7 may besubstituted with other amino acids without affecting the function of theBMP-7 protein.

Among the improved properties of BMP-7 as a result of the abovementioned amino acid substitutions are: increased expression yield,expression in the absence of a pro-domain, increased solubility,increased stability, increased specific biological activity, alteredreceptor specificity, altered receptor binding affinity, alteredco-receptor specificity, altered co-receptor binding affinity, increasedbinding to noggin, reduced binding to noggin and decreasedimmunogenicity,

With respect to the region B amino acids, this means that amino acids atpositions 6, 8, 9, 11, 12, 14, 16-18, 20-31, 35, 36 and 40 of SEQ ID NO:18 may be altered or substituted, without affecting the activity ofBMP-7. In some instances, these amendments (amino acid substitutions),even improved the activity of BMP-7.

The region corresponding to SEQ ID NO: 18 may therefore also bedescribed as shown in formula 1:

Formula 1: (SEQ ID NO: 29) P   K   P   S   S  X1  P  X2 X3  L  X4 X5  I X6  V  X7 X8 X9  D  X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21  M  V   V X22 X23  S   G   S  X24,

wherein;

X1=A or V

X2=T or D or E

X3=Q or D or K or S

X4=N or D or E or H

X5=A or D or S

X6=S or I or L or M or F or Y or E or H or K or Q or R or D

X7=L or E or K or T or M or R

X8=Y or A or D or E or H or K or S or T

X9=F or H or A or D or E or K or Q or R or Y

X10=D or I or L or E or N or S or T

X11=S or D or E or N or R

X12=S or D or E or K or N or T

X13=N or E or Q or R

X14=V or A or D or N or R or T or M or Y or H

X15=I or A or D or E or K or N or Q or R or S or T or V

X16=L or A or E or K or Q

X17=K or D or E or G or Q or R or W

X18=K or A or D or E or H or N or P or Q or S or T or I or V or M

X19=Y or D or E or H or K or Q or I or R

X20=R or D or E or K or N or S

X21=N or D

X22=R or E or S or D or K or Q or L

X23=A or E or D or S

X24=H or R.

The above peptides according to formula 1 may be regarded as equivalentsor variants or analogues of the peptide according to SEQ ID NO: 18. Inone embodiment of the invention, a variant of SEQ ID NO: 18 comprises24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 amino acid substitution(s) selected from the groupconsisting of substitutions X1, X2, X3, X4, X5, X6, X7, X8, X9, X10,X11, X12, X13, X14, X15, X16, X17, X18, X19, X29, X21, X22, X23 and X24.

The amino acids in bold print and underlined in formula 1 represent theamino acids in the peptides according to SEQ ID NO: 18 (wild typesequence) that should preferably not be changed or substituted. Thepreferred amino acids at positions X1-X24 are underlined. Theserepresent the wild type amino acids at the corresponding positions.

In addition, we tested the region B peptides according to SEQ ID NO:6-17 for their OA chondrocyte phenotype changing properties and foundthat they also increased the expression of genes SOX9 and COL2A1 anddecreased the expression of COX-2 (Table 5).

In addition to the above described region B, we also found that peptidesfrom another region (amino acids 58-85 of the mature BMP-7 protein)could advantageously be used to mimic the favorable actions of BMP-7 inOA. Peptides from that region even synergistically improved the actionsof the peptides from the B region described above. This second region isherein described as region A. Peptides from that region A are therewithuseful in the treatment, amelioration or prevention of osteoarthritis incombination with the peptides from region B.

Peptides from this region A may be described as consisting of an aminoacid sequence of between 12 and 28 amino acids in length comprising anamino acid sequence according to SEQ ID NO: 30 (YSEGESAFPLNS) or avariant thereof according to formula 2 (SEQ ID NO: 31), wherein theamino acid sequence of said peptide is comprised in SEQ ID NO: 32(APEGYAAYYSEGESAFPLNSYMNATNHA) or a variant thereof according to formula3 (SEQ ID NO: 33).

Formula 3: (SEQ ID NO: 33)Z1 Z2 Z3 G Y Z4 A Z5 Y S E G Z6 S Z7 Z8 Z9 L Z10 Z11 Z12 M N A T Z13 H A

wherein

Z1=A or I or L or M or Y or V or E or H or K or Q or R,

Z2=P or Y or M,

Z3=E or R or H or K or N or P or Q or S or T or I or L or M or V,

Z4=A or E or Q or R or S,

Z5=Y or N or D,

Z6=E or A or Q,

Z7=A or D or E or H or K or S,

Z8=F or A or D or E or H or Q or R or S,

Z9=P or M,

Z10=N or A or D or S or T or E or Q or R or I or V,

Z11=S or A or D or E or H or K or N or P or Q or T,

Z12=Y or H or D or G or H or N or R or S or T or wherein

Z13=N or F or W or Y or H or K or R.

The above peptides according to formula 3 may be regarded as equivalentsor variants of the peptide according to SEQ ID NO: 32. In one embodimentof the invention, a variant of SEQ ID NO: 32 comprises 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2 or 1 amino acid substitution selected from the groupconsisting of substitutions Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10,Z11, Z12 and Z13.

Accordingly, variants of the region corresponding to SEQ ID NO: 30 maytherefore also be described as shown in formula 2:

Formula 2: (SEQ ID NO: 31) Y S E G Z6 S Z7 Z8 Z9 L Z10 Z11

wherein

Z6=E or A or Q,

Z7=A or D or E or H or K or S,

Z8=F or A or D or E or H or Q or R or S,

Z9=P or M,

Z10=N or A or D or S or T or E or Q or R or I or V or wherein

Z11=S or A or D or E or H or K or N or P or Q or T.

Examples of suitable peptides from the A region are exemplified in Table4.

Preferred variants may include linear peptides, linear retro inversopeptides, retro-inverso peptides, cyclic peptides, mono-looped peptides,and two-looped peptides. CLIPS technology enables the routine productionof such peptides (http://www.pepscan.com/therapeutics/clips-platform).Two-looped peptides may contain two identical peptides from region A orB as described herein or two different peptides from region A or B orcombinations thereof as described herein. Particularly preferred aretwo-looped peptides with one loop comprising a peptide from the A regionas described herein and an other loop comprising a peptide from the Bregion as described herein.

TABLE 4 Peptides from region A suitable for use in thetreatment of OA in combination with peptides from region B. SEQ ID NO:Amino acid sequence 34 APEGYAAYYSEGESAFPLNS 35 PEGYAAYYSEGESAFPLNSY 36EGYAAYYSEGESAFPLNSYM 37 GYAAYYSEGESAFPLNSYMN 38 YAAYYSEGESAFPLNSYMNA 39AAYYSEGESAFPLNSYMNAT 40 AYYSEGESAFPLNSYMNATN 41 YYSEGESAFPLNSYMNATNH 42YSEGESAFPLNSYMNATNHA 43 YSEGESAFPLNS 44 APEGYAAYYSEGESAFPLNS 45PEGYAAYYSEGESAFPLNS 46 EGYAAYYSEGESAFPLNS 47 GYAAYYSEGESAFPLNS 48YAAYYSEGESAFPLNS 50 AAYYSEGESAFPLNS 51 AYYSEGESAFPLNS 52 YYSEGESAFPLNS53 YSEGESAFPLNS 54 YSEGESAFPLNSY 55 YSEGESAFPLNSYM 56 YSEGESAFPLNSYMN 57YSEGESAFPLNSYMNA 58 YSEGESAFPLNSYMNAT 59 YSEGESAFPLNSYMNATN 60YSEGESAFPLNSYMNATNH 61 YSEGESAFPLNSYMNATNHA 62APEGYAAYYSEGESAFPLNSYMNATNHA 63 PEGYAAYYSEGESAFPLNSYMNATNHA 64EGYAAYYSEGESAFPLNSYMNATNHA

TABLE 1Human BMP-7 spanning consecutive 20-mer peptides with at least 2 aminoacids overlap between peptides and their OA chondrocyte phenotype changingproperties. Peptide sequence SEQ ID IL-6 ADAMTS5 COL10A BAPX1/NKX3.2 ALPRUNX2 STGSKQRSQNRSKTPKNQEA 19 — — — — — — KTPKNQEALRMANVAENSSS 20 — — —— — — SSDQRQACKKHELYVSFRDL 21 — — — — — — DLGWQDWIIAPEGYAAYYCE 22 — — —— — — CEGECAFPLNSYMNATNHAI 23 — — — — — — AIVQTLVHFINPETVPKPCC 24 — — —— — — CCAPTQLNAISVLYFDDSSN 25 — — — — — — SSNVILKKYRNMVVRACGCH 26 — — —— — — [-] denotes the absence of a significant effect or a significantlydecreased expression of chondrogenic marker genes BAPX1/NKX3.2, or asignificantly increased expression of OA phenotypic markers IL-6,ADAMTS5, COL10A, ALP and RUNX2 in OA articular chondrocytes, incomparison to untreated OA articular chondrocytes.

TABLE 2Human BMP-7 derived consecutive 20-mer peptides with cysteine residues substituted toserine and their OA chondrocyte phenotype changing properties. Results are visualizedin FIGS. 2-7. SEQ ID IL- BAPX1/ Peptide sequence No: 6 ADAMTS5 COL10ANKX3.2 ALP RUNX2 FINPETVPKPSSAPTQLNAISVLYFDDSSNVILKKYRNMVVRASGSH  1 — —— — — — FINPETVPKPSSAPTQLNAI  2 — — — — — —   NPETVPKPSSAPTQLNAISV  3 —— — — — —     ETVPKPSSAPTQLNAISVLY  4 — — — — — —      VPKPSSAPTQLNAISVLYFD  5 — — — — — +        PKPSSAPTQLNAISVLYFDD 6 + + + + + +         KPSSAPTQLNAISVLYFDDS  7 + + + + + +          SSAPTQLNAISVLYFDDSSN  8 + + + + + +            APTQLNAISVLYFDDSSNVI  9 + + - + + +              TQLNAISVLYFDDSSNVILK 10 + + - + + +                LNAISVLYFDDSSNVILKKY 11 + + + + + +                  AISVLYFDDSSNVILKKYRN 12 + + + + + +                    SVLYFDDSSNVILKKYRNMV 13 + + + + + +                      LYFDDSSNVILKKYRNMVVR 14 + + + + + —                        FDDSSNVILKKYRNMVVRAS 15 + + + + + —                          DSSNVILKKYRNMVVRASGS 16 + + + + + —                           SSNVILKKYRNMVVRASGSH 17 + + + + + +       PKPSSAPTQLNAISVLYFDDSSNVILKKYRNMVVRASGSH 18 + + + + + +[+] denotes a significantly increased expression of chondrogenic markergene BAPX1/NKX3.2, or a significantly decreased expression of OAphenotypic markers IL-6, ADAMTS5, COL10A, ALP and RUNX2 in OA articularchondrocytes, in comparison to untreated OA articular chondrocytes.[-] denotes the absence of a significant effect or a significantlydecreased expression of chondrogenic marker genes BAPX1/NKX3.2, or asignificantly increased expression of OA phenotypic markers IL-6,ADAMTS5, COL10A, ALP and RUNX2 in OA articular chondrocytes, incomparison to untreated OA articular chondrocytes. A peptide wasconsidered as having OA chondrocyte phenotype suppressive activity whenat least 5 out of 6 markers were positive (+). Those peptides are shownin bold.

TABLE 5 Human BMP-7 derived consecutive 20-mer peptides with cysteineresidues substituted to serine and their OA chondrocyte phenotype changing properties. SEQ ID Peptide sequence No: COX-2 COL2A1 SOX9PKPSSAPTQLNAISVLYFDD  6 + + +  KPSSAPTQLNAISVLYFDDS  7 + + +   SSAPTQLNAISVLYFDDSSN  8 + + +      APTQLNAISVLYFDDSSNVI  9 + + +       TQLNAISVLYFDDSSNVILK 10 + + +          LNAISVLYFDDSSNVILKKY11 + + +            AISVLYFDDSSNVILKKYRN 12 + + +             SVLYFDDSSNVILKKYRNMV 13 + + +               LYFDDSSNVILKKYRNMVVR 14 + + +                 FDDSSNVILKKYRNMVVRAS 15 + + +                   DSSNVILKKYRNMVVRASGS 16 + + +                    SSNVILKKYRNMVVRASGSH 17 + + + [+] denotes asignificantly increased expression of chondrogenic marker genes SOX9 andCOL2A1, or a significantly decreased expression of OA phenotypic markerCOX-2 in OA articular chondrocytes, in comparison to untreated OAarticular chondrocytes.

LEGEND TO THE FIGURES

FIG. 1: BMP-7 rescues OA-associated chondrocyte phenotype.

Osteoarthritis chondrocytes display a typical phenotype that ischaracterized by decreased expression of SOX9, COL2A1 and BAPX1/NKX3.2and an increased expression of COX-2 and IL-6 mRNAs (see left panel).BMP-7 (1 nM) is able to rescue this OA-associated chondrocyte phenotypeby normalizing expression of above genes (left panel).

Functional ALP enzyme activity in cell lysates and PGE2 secretion in theculture medium was also normalized upon BMP-7 treatment of OAchondrocytes (right panel).

Expression of indicated mRNAs was determined by RT-qPCR, relatively tocontrol conditions (normalized for 28S rRNA expression). In graphs,error bars represent mean±SEM, statistical differences were calculatedas compared to healthy condition. *=p<0.05.

FIGS. 2-7: Actions of BMP-7 derived peptides on primary human OAarticular chondrocytes.

The action of 20-mer peptides (10 nM) of SEQ ID NO: 2-17 (peptides with1 or 2 amino acids intervals (18-19 amino acid overlap)) and a randompeptide with a sequence according to SEQ ID NO: 49 on the expression ofthe indicated genes was determined on a validated pool of passage 2 OAhuman articular chondrocytes (n=18) and compared to full-lengthrecombinant BMP-7 (1 nM). Samples were harvested after 24 hours andanalyzed for indicated genes by RT-qPCR (normalized for 28S rRNAexpression and relative to control conditions (no peptide exposure)). Inthe graphs, numbers on the x-axis represent individual peptidesaccording to their SEQ ID NO's, error bars represent mean±SEM,statistical differences were calculated (one-way ANOVA with bonferronicorrection) to control condition. *=p<0.05 lower than control conditionfor ADAMTS5, ALP, COL10A1, RUNX2, and IL-6 and higher than controlcondition for BAPX1/NKX3.2. Random peptide is a peptide according to SEQID NO: 49.

FIG. 8: Diagram showing relative expression of COL2A1, Col10A1, COX-2and RUNX2 in isolated chondrocytes from OA patients in the presence of acontrol peptide, BMP-7 and a peptide according to SEQ ID NO: 43.

FIG. 9: Diagrams showing the relative expression of COL10A1 and MMP13 incartilage explants from OA patients in the presence of a controlpeptide, BMP-7 and a peptide according to SEQ ID NO: 43.

EXAMPLES Example 1: OA Chondrocyte Phenotype Suppressive Actions ofBMP-7 Peptides in the Presence of OA Synovial Fluid

We analysed candidate peptides from region B for their OA phenotypesuppressive actions on primary OA articular chondrocytes in the presenceof 20% (v/v) OA synovial fluid (SF). Data were combined from threeindividual OA chondrocyte isolates (n=3) that were each tested intriplicate. Seeded passage-2 OA chondrocytes were exposed to 100 nM ofpeptide in the absence or presence of OA synovial fluid and after 24hours analysed for mRNA expression of genes IL-6, ADAMTS5, COL10A, ALP,BAPX1 and RUNX2 (corrected for 28S rRNA expression and relative tocontrol conditions (no peptide exposure)).

Results were scored as + or − as shown in the footnotes of the tables.

Example 2: Hypertrophy Suppressive Action of BMP-7 Peptides DuringChondrogenic Differentiation of ATDC5 Cells

ATDC5 cells were differentiated using standard protocols for 8 days inthe presence or absence of 1 nM and 10 nM peptides from region B.Peptides were added at start of differentiation and at every mediumchange (multiple) or only at start of differentiation and not duringevery medium change (single). At day 8 in differentiation, samples wereharvested and analysed for gene expression by RT-qPCR (corrected forb-actin expression and relative to t=0).

Example 3: Evaluation of OA-Suppressive Actions in Ex Vivo and In VivoModels for OA

Peptides for use according to the invention may also be investigated inex vivo testing: Full-thickness cartilage biopsies (3 mm punches) fromfemur condyles from TKA (K&L grade 2-3) may be freshly harvesteddirectly after surgery, randomized per patient and taken into ex vivoculture as described previously. Biopsies from patients may be exposedto selected peptides after twenty-four hours at 0.1, 1, 10 or 100 nM for7 days and medium may be changed daily with fresh peptide.

BMP-7 at 1 nM may be used as positive control and vehicle may be used asnegative control. After exposure, the cartilage biopsies may beprocessed to address major cartilage quality-determining parameters. DNAcontent per wet-weight and GAG content may be determined. ALP enzymeactivity may also be investigated. PGE2 and GAG secretion in the culturemedium may be analyzed. Gene expression may be determined for major OAchondrocyte phenotype marker-genes as described herein.

Local OA-related changes in the biopsies as a result of exposure to thepeptides may be analyzed by (immuno)histochemistry (alcian blue- andimmunostainings for COL2A1, SOX9 and COX-2) to further support thenotion that the peptides for use according to the invention are suitablefor medical use in OA.

Diffusion and localization of the peptides into cartilage biopsies maybe addressed in a similar set-up wherein a biotinylated variant of thepeptides may be used. After culture these biopsies may be processed forhistochemistry. Peptide that is diffused into the cartilage biopsies maybe fluorescently visualized by Streptavidin-Alexafluor 488-mediateddetection. This approach may also aid in visualizing the phenotypiccontext and localization of the peptide by MALDI-IMS and help todiscriminate it from potential endogenous BMP-7 fragments.

Example 4: In Vivo Testing

Amino acid sequences of region-B are 100% homologous between human andmouse. In order to further establish the in vivo activity of thepeptides for use according to the invention, representative peptides maybe tested in a well-accepted model for post-traumatic OA, the DMM model.The medial meniscus may be destabilized in 12 weeks old C57BL/6 mice.One week after DMM induction, peptides may be administeredintra-articularly by twice-weekly injections as described previously.Dose may be based on intra-articular BMP-7 studies in which weeklyinjections of 250 ng BMP-7 in a rat knee joint (in 100 μl) showedfavorable outcomes. As 10 μl can be injected in an OA mouse joint anequivalent amount of 25 ng peptide in this volume may be injected perknee joint. An amount of 2.5 and 0.25 ng peptide may also be tested in 2additional groups to determine the pharmacological potency of thepeptide. Saline injections may be used as controls. The sample size ofthis experiment is advantageously 8 mice per group. Animals may besacrificed at consecutive time points after start of peptide treatment(2, 4, 8 weeks). Knee joints may be processed for (immuno)histochemicalanalyses and OARSI scoring (Safranin-O; modified Pritzker).

Example 5: Treatment of Isolated Chondrocytes from OA Patients

Isolated chondrocytes from OA patients (n=6) were treated with BMP-7 (1nM) or the 12-mer peptide according to SEQ ID NO: 43 (1 nM) for 24 h.Pro-chondrogenic (FIG. 8A) and hypertrophic (FIGS. 8B, 8C and 8D) geneexpression was determined via qRT-PCR and normalized for 28S rRNAlevels. These results confirmed our previous findings that BMP-7 or the12 mer peptide induced an upregulation of pro-chondrogenic genes, suchas Col2a1 (A), and a downregulation of pro-hypertrophic genes, such asCOL10A1, COX-2 and RUNX2 (B, C, D). These results show the BMP-7mimicking bioactivity of the core sequence from the region-A peptide.

Example 6: Treatment of Cartilage Explants

4 mm 2 cartilage explants were taken from non-lesion areas of OApatient's knee articular cartilage (n=5) and randomly assigned todifferent experimental treatment conditions (4 explants per treatmentgroup). After a 24 h equilibration period the explants were treated withBMP-7 (1 nM) or the 12-mer peptide according to SEQ ID NO: 43 (10 nM)for 24 h. Hypertrophic gene expression was determined via qRT-PCR andnormalized for 28S rRNA levels. After treatment with BMP-7 or the 12 merwe observed a downregulation of pro-hypertrophic genes, such as Col10a1(FIG. 9A) and MMP13 (FIG. 9B). These results are in line with theeffects described above and show the BMP-7 mimicking bioactivity of thepeptides according to the invention.

Example 7: Staining of Cartilage Explants

Cartilage explants obtained from 2 patients were cultured for 14 days inthe presence of BMP-7 (1 nM) or BMP-7 mimicking peptideSVLYFDDSSNVILKKYRNMV (SEQ ID NO: 13) at 10 nM. Glycosaminoglycans(GAGs), an important component of the extracellular matrix (ECM), werestained with Safranin-O (in red) and other tissues are counterstainedwith Fast green (in green/blue).

Both patients showed an increased Safranin-O intensity in BMP7 andpeptide SVLYFDDSSNVILKKYRNMV (SEQ ID NO: 13) treated explants comparedto control.

These results are in line with the effects described above and show theBMP-7 mimicking bioactivity of the peptides according to the invention.

Example 8: Treatment of Cartilage Explants

Cartilage explants of 3 mm² were taken from OA patient's knee cartilage(n=6) and treated with BMP-7 (1 nM), with peptide SVLYFDDSSNVILKKYRNMV(SEQ ID NO: 13) at 10 nM or a scrambled irrelevant control peptide (10nM) for 14 days. Prostaglandin E2 (PGE2) levels, a pro-hypertrophicfactor, were determined via an ELISA.

Synovial tissue samples from OA patients (n=6) were treated with BMP-7(1 nM), with peptide SVLYFDDSSNVILKKYRNMV (SEQ ID NO: 13) at 10 nM or ascrambled irrelevant control peptide (10 nM) for 24 h. PGE2 levels weredetermined via an ELISA.

Hoffa's fat pad tissue samples from OA patients (n=6) were treated withBMP-7 (1 nM) with peptide SVLYFDDSSNVILKKYRNMV (SEQ ID NO: 13) at 10 nMor a scrambled irrelevant control peptide (10 nM) for 24 h. PGE2 levelswere determined via an ELISA.

Meniscus tissue samples from OA patients (n=6) were treated with BMP-7(1 nM) with peptide peptide SVLYFDDSSNVILKKYRNMV (SEQ ID NO: 13) at 10nM or a scrambled irrelevant control peptide (10 nM) for 24 h. PGE2levels were determined via an ELISA.

The results unequivocally showed reduce PGE2 levels in the cartilage andthe surrounding tissues after BMP-7 or peptide SVLYFDDSSNVILKKYRNMV (SEQID NO: 13) treatment. Treatment with the scrambled peptide did notresult in a reduction of PGE2 levels.

These results are in line with the effects described above and show theBMP-7 mimicking bioactivity of the peptides according to the invention.

REFERENCES

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1. A method of treating subject at risk of developing osteoarthritis,the method comprising: administering to the subject a peptide; whereinthe peptide consists of an amino acid sequence according to SEQ ID NO:18 or an analogue thereof, wherein the analogue is a peptide consistingof an amino acid sequence according to formula 1 (SEQ ID NO: 29), or afragment thereof; wherein the fragment consists of at least 10consecutive amino acids of SEQ ID NO: 18 or an amino acid sequenceaccording to formula 1, wherein formula 1 is: (SEQ ID NO: 29)P K P S S X1 P X2 X3 L X4 X5 I X6 V X7 X8 X9 D X10X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21 M V V X22 X23 S G S X24,

and wherein; X1=A or V X2=T or D or E X3=Q or D or K or S X4=N or D or Eor H X5=A or D or S X6=S or I or L or M or F or Y or E or H or K or Q orR or D X7=L or E or K or T or M or R X8=Y or A or D or E or H or K or Sor T X9=F or H or A or D or E or K or Q or R or Y X10=D or I or L or Eor N or S or T X11=S or D or E or N or R X12=S or D or E or K or N or TX13=N or E or Q or R X14=V or A or D or N or R or T or M or Y or H X15=Ior A or D or E or K or N or Q or R or S or T or V X16=L or A or E or Kor Q X17=K or D or E or G or Q or R or W X18=K or A or D or E or H or Nor P or Q or S or T or I or V or M X19=Y or D or E or H or K or Q or Ior R X20=R or D or E or K or N or S X21=N or D X22=R or E or S or D or Kor Q or L X23=A or E or D or S X24=H or R; wherein the fragmentcomprises Leu at a position c or responding to position 16 of SEQ ID NO:29 or Ser at a position corresponding to position 37 of SEQ ID NO: 29.2. The method according to claim 1, wherein the analogue of SEQ ID NO:18 comprises 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution(s) selected from thegroup consisting of substitutions X1, X2, X3, X4, X5, X6, X7, X8, X9,X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X29, X21, X22, X23,and X24.
 3. The method according to claim 1, wherein the peptide isselected from the group consisting of linear peptides, linearretro-inverso peptides, retro-inverso peptides, cyclic peptides,mono-looped peptides, and two-looped peptides.
 4. The method accordingto claim 1, wherein the treatment further comprises the administrationof at least one polypeptide, wherein the polypeptide is between 12 and28 amino acids in length and comprises an amino acid sequence accordingto SEQ ID NO: 30 or a variant thereof according to (SEQ ID NO: 31, andwherein the amino acid sequence of the polypeptide is comprised in SEQID NO: 32 or a variant thereof according to SEQ ID NO:
 33. 5. The methodaccording to claim 4, wherein the variant according to SEQ ID NO: 33comprises 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitution(s) selected from the group consisting of substitutions Z1,Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, Z12 and Z13, wherein; Z1=A orI or L or M or Y or V or E or H or K or Q or R, Z2=P or Y or M, Z3=E orR or H or K or N or P or Q or S or T or I or L or M or V, Z4=A or E or Qor R or S, Z5=Y or N or D, Z6=E or A or Q, Z7=A or D or E or H or K orS, Z8=F or A or D or E or H or Q or R or S, Z9=P or M, Z10=N or A or Dor S or T or E or Q or R or I or V, Z11=S or A or D or E or H or K or Nor P or Q or T, Z12=Y or H or D or G or H or N or R or S or T or whereinZ13=N or F or W or Y or H or K or R.
 6. The method according to claim 4,wherein the variant according to SEQ ID NO: 31 comprises 5, 4, 3, 2 or 1amino acid substitution(s) selected from the group consisting ofsubstitutions Z6, Z7, Z8, Z9, Z10 and Z11, wherein Z6=E or A or Q, Z7=Aor D or E or H or K or S, Z8=F or A or D or E or H or Q or R or S, Z9=Por M, Z10=N or A or D or S or T or E or Q or R or I or V or whereinZ11=S or A or D or E or H or K or N or P or Q or T.
 7. The methodaccording to claim 4, wherein the polypeptide comprises an amino acidsequence according to SEQ ID NO:
 30. 8. The method according to claim 4,wherein the amino acid sequence of the polypeptide is fully comprised inSEQ ID NO:
 32. 9. The method according to any one of claims 4-8 whereinthe polypeptide is selected from the group consisting of linearpeptides, linear retro-inverso peptides, retro-inverso peptides, cyclicpeptides, mono-looped peptides, and two-looped peptides.
 10. The methodaccording to claim 4, wherein the polypeptide consists of an amino acidsequence selected from the group consisting of SEQ ID NOs: 34-48 and SEQID NOs: 50-64.
 11. The method according to claim 1, wherein the subjectsuffers from at least one of synovial inflammation, synovial fibrosis,previous joint injury, abnormal joint or limb development, abnormalalignment of joints, one leg of a different length, high levels of jointstress, and cartilage loss.